Internal death programs play significant roles in many diseases. In this project, we are taking a multifaceted approach to studying molecular mechanisms of both apoptotic and nonapoptotic death programs in lymphocytes as well as other cell types. We have discovered that inhibition of caspase-8 in non-lymphoid cells can lead to a form of cell death exhibiting particular cytoplasmic double membrane structures called autophagy. Autophagy is a process evolutionarily conserved from humans to yeast, by which cytoplasmic proteins and organelles are catabolized. An understanding is just emerging about how cells select between autophagic cell death and survival. Mitochondria have a primary physiological role in producing ATP as an energy source, but also regulate cell death. In response to cellular stress, dysfunctional mitochondria produce ROS and other pro-death mediators to initiate programmed cell death pathways, including apoptosis or necroptosis. Mitophagy, a selective form of autophagy, can target dysfunctional mitochondria for lysosomal degradation and protect cells from oxidative damage and necrosis. This is beneficial for the survival of terminally differentiated cells, like nerve and heart muscle cells. When mitophagy fails then cellular degeneration can occur. Several regulators of mitophagy, including PINK1, Nix (BNIP3L), and PARKIN have been identified. Mutations or deletions of those genes have been associated with a various diseases, including ischemic injury in myocardial infarction and stroke, as well as neurodegenerative disease. Hence, understanding the detailed mechanism of mitophagy remains an important goal for improving the diagnosis and treatment of diseases involving mitochondria. Two autosomal recessive Parkinson's disease genes, PINK1 (PTEN induced putative kinase 1) and PARKIN, regulate mitophagic clearance of dysfunctional mitochondria. In healthy cells, PINK1 is constitutively degraded by mitochondrial proteases, such as the mitochondria inner membrane protease Presenilin Associated, Rhomboid-Like (PARL) protein. Membrane depolarization of dysfunctional mitochondria inhibits PINK1 degradation, causing it to accumulate and promote mitophagy via recruitment of another familial Parkinson's protein, the E3 ubiquitin ligase PARKIN. However, the detailed mechanism of PINK1 degradation and stabilization remains unclear. We have studied PGAM5, paralog member 5 of a family of highly conserved phosphoglycerate mutases, which is a 32-kDa mitochondrial protein that apparently lacks phosphotransfer function on phosphoglycerates, but retains activity as a serine/threonine protein phosphatase that regulates the ASK1 kinase. Thus, it is important to establish the in vivo role of PGAM5 on mitochondria. Using a new strain of knockout mice, we found that PGAM5 is critical for PINK1 resistance to PARL degradation and its stabilization on damaged mitochondria to initiate mitophagy. Loss of PGAM5 totally disables PINK1 stabilization and impairs mitophagy. Cells deficient with PGAM5 showed elevated ROS originated from mitochondria, and exacerbated cell necrosis compared to control wild type cells. This is likely due to poor mitophagy and the persistence of damaged or dysfunctional mitochondria. Necroptosis as a molecular program, rather than simply incidental cell death, was established by elucidating the roles of receptor interacting protein (RIP) kinases 1 and 3, along with their downstream partner, mixed lineage kinase-like domain protein (MLKL). Previous studies suggested that phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein that associates with RIP1/RIP3/MLKL complex, promotes necroptosis. We have generated mice deficient in the pgam5 gene and surprisingly found PGAM5-deficiency exacerbated rather than reduced necroptosis in response to multiple in vitro and in vivo necroptotic stimuli. For example, PGAM5-deficiency worsens ischemic reperfusion injury (I/R) in the heart and brain. Electron microscopy, biochemical, and confocal analysis revealed that PGAM5 is indispensable for the process of PINK1-dependent mitophagy which antagonizes necroptosis. The loss of PGAM5/PINK1 mediated mitophagy causes the accumulation of abnormal mitochondria, leading to the overproduction of reactive oxygen species (ROS) that promotes necroptosis. Our results revise the former proposal that PGAM5 acts downstream of RIP1/RIP3 to mediate necroptosis. Instead, PGAM5 protects cells from necroptosis by independently promoting mitophagy. PGAM5 promotion of mitophagy may represent a therapeutic target for stroke, myocardial infarction and other diseases caused by oxidative damage and necroptosis. Taken together, our data suggest that PGAM5 promotes PINK1-mediated mitophagy, which could be cytoprotective in ischemic injuries. Our studies have shed new light on the mechanism of PINK1 stabilization by unveiling a new function of the mitochondrial regulatory protein PGAM5. PINK1 stabilization and subsequent parkin recruitment triggers mitophagy, which selectively eliminates dysfunctional mitochondria to protect cells/tissues from oxidative stress and cell death. in our investigations, we have generated new evidence suggesting that mitophagy may contribute to dopaminergic neurodegeneration and movement disorders in experimental animals by the role of PGAM5 in these processes. Contrary to a previous study in drosophila, in a mammalian system we found PGAM5 protects dopamine neurons from degeneration, presumably by promoting PINK1 stabilization. Consistent with a mitochondrial pathogenesis for Parkinsons disease, PINK1 deficiency in drosophila causes energy depletion, shortened lifespan, and dopamine neuron degeneration. Aged PINK1 deficient mice show impaired neural activity similar to PGAM5 KO mice, but without DA neurodegeneration; and mutations in PINK1 and parkin predispose to the movement disorders and DA neurodegeneration that is characteristic of familial Parkinsons disease in humans. The new role for the mitochondrial protein PGAM5 in dopamine neuron pathology, lends further weight to the mitochondrial theory of Parkinson's pathogenesis that has been developed by Dr. Richard Youle in NINDS.